Methods for the isolation of proteinase inhibitor from potato tubers

ABSTRACT

A rapid and simple method of isolating heat stable proteinase inhibitor proteins from plant tissues such as potato tubers is disclosed. The method comprises three steps. Proteins from potato tubers are extracted in an aqueous/alcohol extraction medium to form an alcohol extract. The alcohol extract is heated to a first temperature then cooled to a second temperature to form an insoluble precipitate phase containing debris and a soluble phase that contains the heat stable proteinase inhibitor proteins. The heat stable proteinase inhibitor proteins are precipitated from the soluble phase by dialysis against a suitable dialysis medium. The precipitated proteins may include a single inhibitor protein, or a mixture thereof.

TECHNICAL FIELD

[0001] This invention is generally directed to methods for the isolationof proteinase inhibitor proteins from plant tissues and, moreparticularly, to the isolation of heat stable proteinase inhibitorproteins from potato tubers.

BACKGROUND OF THE INVENTION

[0002] Tubers from Solanaceous plants such as potato, are an abundantsource of a wide range of classes of proteinase inhibitor proteins. Manyof these proteins inhibit the activity of digestive proteinases, such astrypsin and chymotrypsin that naturally occur in both insects andmammals. By disturbing the natural digestive process, proteinaseinhibitors form part of a plant's natural defense against foraging byherbivores. For the same reason proteinase inhibitors have applicationin the pest control industry for the control of insects such as fireants. In addition, because they also inhibit human digestiveproteinases, these inhibitors have value in the pharmaceutical industryfor the control of obesity and diabetes.

[0003] Potato tubers have been a major source for the study andpreparation of proteinase inhibitors. Several of the proteinaseinhibitors present in potato tubers are of the heat stable variety.Among these are two Kunitz-type proteinase inhibitors having a subunitMr of about 20 and 20.5 kD that inhibit both trypsin and chymotrypsin(Walsh and Twichell, Plant Physiol. 97: 15-18, 1991). The smallerprotein is a powerful inhibitor of chymotrypsin, and a weak inhibitor oftrypsin. The larger protein is a powerful inhibitor of trypsin. Otherheat stable proteinase inhibitors include one with a subunit Mr of about9.5 kD (designated proteinase inhibitor I) which is a strong inhibitorof chymotrypsin (Melville and Ryan, J. Biol. Chem. 247:3445-3453, 1972),and another with a subunit Mr of about 10.5 kD (designated proteinaseinhibitor II) which is a strong inhibitor of both chymotrypsin andtrypsin (Bryant, Green and Ryan,. Biochemistry 15: 3418-3424, 1976).

[0004] Previous methods for preparation of these heat stable proteinaseinhibitors have incorporated numerous steps, including: extraction inthe presence of dithionate, heat, ammonium sulfate precipitation andchromatography (Walsh and Twichell, Plant Physiol. 97: 15-18, 1991;Melville and Ryan, J. Biol. Chem. 247: 3445-3453, 1972; Bryant, Greenand Ryan, Biochemistry 15: 3418-3424, 1976; Ryan and Kassell, Methods inEnz. XIX: 883-889, 1970; Pearce and Ryan, Anal. Biochem. 130: 223-225,1983). Unfortunately these methods are cumbersome, tedious, timeconsuming, expensive and produce relatively low yields of heat stableproteinase inhibitors. A major problem is that initial extracts form apasty homogenate with poor flow characteristics resulting indifficulties with subsequent processing steps, especially filtration,ammonium sulfate precipitation and resolubilization. The pastyconsistency sometimes requires the use of pressurized filtration duringsubsequent steps, especially in larger scale extractions (Ryan andKassell, 1970). Furthermore, the pasty consistency reduces yield becauseof difficulty in fully recovering material from the paste. Theseprevious methods are also disadvantageous because they require skilledlabor to execute and often take several days to complete.

[0005] Accordingly, there is a need in the art for a method of isolatingproteinase inhibitors from plant tissue, especially potato tubers, thatovercomes the problem of the pasty extract, eliminates the need forammonium sulfate precipitation, is rapid, inexpensive, simple to performand easy to accomplish on any scale from the laboratory to a largeindustrial process. The present invention fulfills these needs andprovides other related advantages.

SUMMARY OF THE INVENTION

[0006] In brief, this invention relates to rapid and simple methods ofisolating heat stable proteinase inhibitor proteins from plant tissuescontaining the same, particularly potato tubers. The method comprisesthree steps. First, proteins from potato tubers are extracted in solubleform in an aqueous/alcohol extraction medium such as dilute formic acidand 20% ethanol. The presence of the ethanol in the extraction mediumconverts the otherwise pasty homogenate to a smooth flowing alcoholextract that is easy to manipulate and particularly easy to filter.Second, the alcohol is heated to a first temperature then cooled to asecond temperature. Heating in the presence of alcohol denatures most ofthe unwanted proteins in the alcohol extract, and subsequent coolingleads to formation of a precipitate phase constituting debris and asoluble phase that contains the heat stable proteinase inhibitorproteins. Third, the heat stable proteinase inhibitor proteins areprecipitated from the soluble phase by dialysis against a suitabledialysis medium, such as dilute formic acid or water followed by diluteformic acid. The precipitated proteins may be either a single inhibitorprotein—proteinase inhibitor II, or a mixture of proteinase inhibitor IIand two Kunitz proteinase inhibitors (one most active against trypsinand the other most active against chymotrypsin). The precipitatedproteinase inhibitor proteins are free of the bulk of other proteins andother constituents originally present in the tuber.

[0007] Whether the precipitated proteins constitute the single inhibitoror the mixture of inhibitors is determined by a single modification.This modification relates to the heating temperature selected for thedenaturation step. The single inhibitor may be obtained upon heating to70° C., cooling to 50° C. followed by dialysis against 0.22% formicacid. The inhibitor mixture may be obtained by heating to 50° C.,cooling to room temperature followed by dialysis against water, wherethe water dialysis is further followed by the addition formic acid to0.88%.

[0008] Excluding dialysis time, the entire method can be performed inless than an hour compared with the days that were required for previousmethods. The method is simple, can be performed by unskilled techniciansand is amenable to performance on any scale. It avoids the use ofexpensive ammonium sulfate and chromatography steps, and even theethanol can be recovered which further reduces the process costs of themethod. The method can yield 300 mg of substantially pure proteinaseinhibitor proteins from 1 pound of potatoes which is vastly superior tothe 150 mg yield of less pure proteins from 100 pounds of potatoes usingprior methods.

[0009] These and other aspects of the invention are evident uponreference to the following detailed description and Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 is the HPLC analysis of a representative heat-treatedaqueous/alcohol extract prepared according to this invention beforedialysis.

[0011]FIG. 2 is the HPLC analysis of a first, post-dialysis, proteinaseinhibitor protein mixture prepared according to this invention.

[0012]FIG. 3 illustrates gel electrophoresis analysis of a firstproteinase inhibitor protein mixture prepared according to thisinvention.

[0013]FIG. 4 illustrates proteinase inhibitor activity of a firstproteinase inhibitor protein mixture prepared according to thisinvention.

[0014]FIG. 5 illustrates proteinase inhibitor activity of a secondproteinase inhibitor protein mixture prepared according to thisinvention.

[0015]FIG. 6 is the HPLC analysis of a second proteinase inhibitorprotein mixture prepared according to this invention

[0016]FIG. 7 illustrates gel electrophoresis analysis of a secondproteinase inhibitor protein mixture prepared according to thisinvention

DETAILED DESCRIPTION OF THE INVENTION

[0017] As mentioned above, the present invention is directed to theisolation of one or more heat stable proteinase inhibitor proteins froma plant tissue which contains the same. Potato tubers are a particularlywell studied plant tissue which are known to abundantly contain severalclasses of heat stable proteinase inhibitors. Therefore, in oneembodiment of this invention, a method is disclosed for isolation ofheat stable proteinase inhibitor proteins from potato tubers. It willalso be evident to those skilled in the art that this method can beadapted to the extraction of other proteinase inhibitors from otherplant tissues, provided the inhibitors are heat stable and soluble in asolution comprising alcohol and water. In addition to potato tubers,representative tissues may include but are not be limited to, tubers ofsweet potatoes or cassava, leaves and fruits of potatoes, tomatoes andlegumes, seeds of all plant species, as well as genetically engineeredorganisms expressing proteinase inhibitor genes.

[0018] In the practice of this invention, the method for preparingproteinase inhibitor proteins from plant tissue includes three steps.

[0019] In the first step, proteins are extracted from the tissue in anaqueous/alcohol extraction medium to obtain an alcohol extract.“Extraction” means to disrupt cellular structure of the plant tissue soas to release and solubilize cellular contents in the extraction medium.The initial step of extraction is commonly known in the art as“homogenization”, which may comprise a disruptive mechanical processsuch as crushing, grinding, blending, sonicating or other technique. Inthe present invention the extraction medium is comprised of a solutionhaving an aqueous portion and an alcohol portion. The aqueous portionideally should be selected to be one that will preferentially solubilizeproteinase inhibitors while simultaneously promoting precipitation ofunwanted proteins and other macromolecules released from the tissue.Acidic solutions and solutions containing high salt (greater than 0.3M), have a tendency to precipitate many macromolecules, such as proteinsand nucleic acids, whereas several proteinase inhibitors are soluble inthe same. Therefore, an embodiment of this invention uses an aqueousportion comprised of dilute formic acid and high salt. Alcohol also hasa tendency to precipitate macromolecules and this invention disclosesthat some proteinase inhibitors remain soluble in solutions containingalcohol. The extraction medium therefore also includes an alcoholportion. The alcohol may be added to the aqueous portion of theextraction medium during or after homogenization of the plant tissuewithout substantially altering results. In the embodiments disclosedherein, alcohol is added to the aqueous solution during thehomogenization process.

[0020] Ethanol is used at about 20% v/v in the preferred embodimentsdescribed herein. However, other alcohols, including but not limited to,methanol, propanol or phenol may be substituted as well as other watermiscible organics. Substitute alcohol(s) may be evaluated by extractinga plant tissue using mediums comprising a variety of alcohols at avariety of concentrations. After centrifuging the debris, measurement ofprotein levels and specific proteinase inhibitor activities in thesupernatant determines an alcohol type and concentration that yields thehighest specific activity.

[0021] The initial homogenate may be prepared in a solution of diluteformic acid and salt. For example, potato tubers may be homogenized in ablender in a medium having a final concentration of about 0.2% formicacid and 0.3 M NaCl. Ethanol is added during the homogenization process.Addition of ethanol in this manner is particularly useful for extractsprepared from potato tubers because the ethanol changes the consistencyof the homogenate from a thick starchy paste to a smooth flowing alcoholextract. This facilitates subsequent manipulations of the alcoholextract, such as removal of coarse debris. In the embodiments describedherein, debris is removed by squeezing the alcohol extract throughcheesecloth. Other ways to accomplish removal of debris include, but arenot limited to, centrifugation, filtration through natural materialslike diatomaceous earth, filtration through synthetic filters, extrusionthrough screen meshes and other techniques.

[0022] In the second step, the alcohol extract is heated to a firsttemperature then cooled to a second temperature. This causesdenaturation and precipitation of many unwanted proteins present in theextract and forms an insoluble precipitate phase and a soluble phase.Denaturation is a process that disrupts secondary and tertiarystructures of proteins causing them to unfold or otherwise lose theirnative characteristics. Denaturation may be irreversible if the proteinscannot afterward be made to regain their native characteristics. Alcoholpromotes the denaturation of many proteins, especially in the presenceof heat. Upon cooling, denatured proteins are susceptible to aggregateto form insoluble precipitates. In the practice of this invention,several proteinase inhibitors are not irreversibly denatured and remainsoluble when heated and cooled in the presence of alcohol, while mostother proteins precipitate. In particular, the embodiments describedherein show that several proteinase inhibitors from potato tuber remainactive and soluble in an alcohol extract containing 20% ethanol that hasbeen heated to a first temperature and cooled to a second temperature.

[0023] Generally, the alcohol extract should be heated to a firsttemperature that is hot enough to denature unwanted proteins, but not-sohot as to irreversibly denature the proteinase inhibitors The extractshould then be cooled to a second temperature low enough to facilitateaggregation of a precipitate. After cooling, an insoluble precipitatephase containing denatured proteins and other debris will develop. Theprecipitate phase can be separated from the remaining soluble phase bycentrifugation, filtration or other equivalent method to obtain a clearsoluble phase. The soluble phase contains proteinase inhibitor protein.

[0024] Selection of the first and second temperatures of the heatingstep can be determinative of the type of inhibitor obtained becausedifferent proteinase inhibitors have different denaturation and/orprecipitation properties. In one embodiment using alcohol extracts ofpotato tubers, the first temperature is 70° C. and the secondtemperature is 50° C. This yields a preparation containing proteinaseinhibitor II. In another embodiment using the same alcohol extract ofpotato tubers, the first temperature is 50° C. and the secondtemperature is room temperature. This yields a mixture containing threedifferent inhibitors—proteinase inhibitor II and two Kunitz proteinaseinhibitors, one most active against trypsin and the other most activeagainst chymotrypsin.

[0025] The method allows selection of the first and second temperaturesfor the heating step to be modified as needed to obtain best yields ofparticular types of proteinase inhibitor proteins and for particularvarieties of plant tissue. For example, within one embodiment, alcoholextracts from a particular plant tissue can be treated to a series ofdifferent first and second temperatures. After centrifuging the debris,measurement of protein levels and specific proteinase inhibitoractivities in the supernatant determines the heat treatment that yieldsthe highest specific activity.

[0026] The alcohol can be recovered from the soluble phase after heattreating the alcohol extract. In one embodiment, the ethanol may berecovered by evaporation at the second temperature to which the alcoholextract has been cooled. In another embodiment, the alcohol need not berecovered. In either case, the presence or absence of the alcohol willnot effect subsequent steps in the method.

[0027] In the third step of the method, proteinase inhibitors arerecovered from the soluble phase by precipitation via dialysis against amedium that will promote precipitation of the inhibitors, preferably anacid dialysis solution employing a protic acid, such as an organic acid,and more preferably formic acid. The dialysis not only promotesprecipitation for recovery of the proteins but also eliminates smallerproteins and other molecules carried over from previous steps. Selectionof the dialysis medium is, in part, determined by the proteinaseinhibitors to be isolated. In one embodiment, dilute formic acid atabout 0.22% is used to recover of proteinase inhibitor II from potatotubers. In another embodiment, tap water is preferred to recover amixture of three proteinase inhibitors from potato tubers. When tapwater is used as the dialysis medium the proteins may be precipitated bythe subsequent addition of formic acid to about 0.88%. In either case,the dialysis is across a membrane having a molecular weight cutoff of12,000-14,000 Daltons. As the salt, residual alcohol and other smallmolecules present in the soluble extract are removed, a whiteprecipitate will form containing the proteinase inhibitors. In alaboratory scale embodiment, twelve to 24 hours of dialysis issufficient for complete precipitate formation. However, other methods ofdialysis, solute exchange, or precipitation that may yield a better ormore rapid recovery of the proteinase inhibitors can be substituted.

[0028] The precipitated proteins may be recovered by filtration,centrifugation or other methods. The precipitated proteins contain amixture of proteinase inhibitor proteins of a variety of degrees ofpurity. One or more proteinase inhibitor proteins may constitute >90% ofthe total protein in the precipitate. Such a precipitate may beconsidered a substantially pure mixture of proteinase inhibitorproteins. The precipitate can be dissolved in a solvent suitable for anintended use or subsequent formulation of the proteinase inhibitors. Inthe embodiments presented herein, the precipitates are dissolved in 0.1M ammonium bicarbonate which is suitable for solubilization andsubsequent lyophilization—a known method for stable storage ofproteinase inhibitors.

[0029] The general methodology described herein is suitable for thepreparation of a variety of heat stable proteinase inhibitor proteinsfrom a variety of plant tissue provided that the inhibitors areresistant to irreversible denaturation in the presence of alcohol andheat. This method yields mixtures of proteinase inhibitor proteinshaving various degrees of purity depending upon the specific tissueused, proteinase inhibitors present and on the particular alcohols,temperatures and dialysis medium employed for the method. When optimizedfor a given tissue, high yields of substantially pure compositions ofproteinase inhibitors can be obtained as shown by the examples forpotato tuber proteinase inhibitors described below. To this end, thefollowing examples are presented for purposes of illustration, notlimitation.

EXAMPLES Example 1 Preparation of Potato Proteinase Inhibitor II

[0030] One lb. of Russet Burbank potatoes were homogenized in a blenderin the presence of 100 ml 0.88% formic acid and 1.5 M NaCl for 2minutes. While blending, 125 ml of 95% ethanol was slowly added to thehomogenate to bring the final ethanol concentration to about 20%. Thischanged the homogenate from a paste to a liquid mixture whichfacilitates filtration. Coarse insoluble debris were removed bysqueezing the liquid through 8 layers of cheesecloth. The liquidfiltrate was collected as the alcohol extract.

[0031] The alcohol extract was transferred to an evaporator flask andheated with stirring by immersing the flask in a boiling water bathuntil the temperature reached 70° C. At that point the flask wasattached to flash evaporator with a bath temperature at 50° C. Theevaporator was used to recover the ethanol, however, it was notnecessary to remove the ethanol to continue with the method. Theresulting liquid was centrifuged at 4000× g to remove excess starch andthe precipitated insoluble phase which formed, the clear supernatantrepresenting the soluble phase was collected.

[0032] The soluble phase was placed in a 12,000-14,000 molecular weightcutoff dialysis band and dialyzed against 10 liters of 0.22% formic acidfor at least 12 hours with several changes of the formic acid solution.By this time a white precipitate formed in the bag. The precipitate waspure proteinase inhibitor II which was recovered by centrifugation at4000× g for 5 minutes. The precipitate was dissolved in 0.1 M ammoniumbicarbonate and lyophilized. The yield was approximately 57 mg inhibitorper lb. potatoes. Better yields may be achieved by starting with newlydug potatoes or potato cultivars having higher levels of proteinaseinhibitor II.

[0033]FIGS. 1 and 2 show HPLC analyses of the materials recovered fromthis procedure. Both figures show spectrophotometeric absorbance at 280nm of materials eluted from a semipreparative C18 column (Vydac, Catalog#218TP510, 5 10×250 mm) which was developed at 2 ml/min over 45 minuteswith a linear gradient from 20%.solvent A (0.1% trifluoroacetic acid) to50% solvent B (0.1% trifluoroacetic/acetonitrile). FIG. 1 shows thesoluble material present in the clear soluble phase recovered after heatdenaturation of the alcohol extract in step 2 before dialysis. FIG. 2shows the material present in the precipitate formed after dialysis instep 3. The precipitate was dissolved in solvent A for analysis.

[0034]FIG. 3 shows a gel electrophoresis analysis of the material inpeak #1 from the HPLC analysis of FIG. 2. Lane 1 is a sample of theinitial potato homogenate prior to the addition of ethanol. Lanes 2, 3,and 4 are samples of the leading, middle, and trailing fractions,respectively, of peak #1. Peak 1 was further subject to immunologicalanalyses which identified the material as potato proteinase inhibitorII.

[0035]FIG. 4 shows proteinase inhibitor II activity of peak #1 from FIG.2. The material was shown to be a strong inhibitor of both trypsin (T)and chymotrypsin (C). For trypsin inhibition, 4.0 μg of the materialinhibited 2 μg of trypsin. For chymotrypsin inhibition, 1.4 μginhibited. 1.5 μg of chymotrypsin. This double-headed inhibitor activityis characteristic of proteinase inhibitor II.

Example 2 Preparation of a Mixture of Potato Proteinase Inhibitors fromPotato

[0036] An alcohol extract was prepared as described above in Example 1.The alcohol extract was heated with stirring by immersing the flask in aboiling water bath until the temperature reached 50° C. At that pointthe flask was immersed in a cold running water bath so that thetemperature was reduced to room temperature. The resulting liquid wascentrifuged at 4000× g to remove excess starch and the precipitatedinsoluble phase, the clear supernatant representing the soluble phasewas collected.

[0037] The soluble phase was placed in a 12,000-14,000 molecular weightcutoff dialysis band and dialyzed against running tap water for 24hours. After this time either the solution in the bag was adjusted to0.88% formic acid or alternatively the dialysis solution was adjusted to0.88% formic acid and dialysis was continued for an additional 4 hours.By this time a white precipitate formed in the bag. The precipitatecontained a mixture of proteinase inhibitor II and two Kunitz familyproteinase inhibitors as shown in the FIGS. 5 and 6 below. Theprecipitate was recovered, dissolved and lyophilized as in Example 1.The yield was approximately 300 mg inhibitors per lb. potatoes. Again,better yields may be possible by starting with newly dug potatoes orpotato cultivars having higher levels of proteinase inhibitor II.

[0038]FIG. 5 shows the proteinase inhibitor activity of the recoveredprecipitated mixture. The material was found to strongly inhibit bothtrypsin (T) and chymotrypsin (C). For trypsin inhibition, 3.2 μg of thematerial inhibited 2.0 μg of trypsin. For chymotrypsin inhibition, 2.2μg inhibited 1.5 μg of chymotrypsin.

[0039]FIG. 6 shows HPLC analysis of the final precipitated materialrecovered from this procedure. Conditions were the same as described inFIG. 2. The three identified peaks were subject to gel electrophoresisanalysis as shown in FIG. 7. Lanes 1, 2, and 3 are the materials frompeaks #1, #2, and #3, respectively, of FIG. 6. As before, immunologicalanalyses identified the material in peak #1 as potato proteinaseinhibitor II. The materials from peaks 2 and 3 were subjected to aminoacid sequence analysis. The analysis identified peak 2 as aKunitz-family inhibitor having a powerful chymotrypsin inhibitoryactivity and a weak trypsin inhibitor. Peak 3 was identified asKunitz-family inhibitor having a powerful trypsin inhibitor activity.

[0040] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not to be limited except as by the appended claims.

We claim:
 1. A method for obtaining a heat stable proteinase inhibitor protein from plant tissues containing the same, comprising the steps of: extracting the proteinase inhibitor protein from the plant tissues in an aqueous/alcohol solvent to form an alcohol extract containing a solubilized proteinase inhibitor protein; heating the alcohol extract to a first temperature followed by cooling to a second temperature to form an insoluble precipitate phase and a soluble phase; and precipitating the proteinase inhibitor protein from the soluble phase by dialysis against an acidic dialysis solution.
 2. The method of claim 1 wherein the plant tissues are potato tubers.
 3. The method of claim 1 wherein the proteinase inhibitor protein is potato proteinase inhibitor II.
 4. The method of claim 1 wherein the proteinase inhibitor protein is a mixture of potato proteinase inhibitor II, a Kunitz-family chymotrypsin/trypsin inhibitor having a dominant chymotrypsin inhibitory activity, and a Kunitz-family trypsin/chymotrypsin inhibitor having a dominant trypsin inhibitory activity.
 5. The method of claim 1 wherein the aqueous/alcohol solvent is comprised of a final concentration of about 0.2% formic acid, about 0.3 M NaCl and about 20% ethanol; the first temperature is about 70° C. or above; the second temperature is about 50° C. or below; and the aqueous dialysis solution contains about 0.22% formic acid.
 6. The method of claim 1 wherein the aqueous/alcohol solvent comprises of about 0.2% formic acid, about 0.3 M NaCl and about 20% ethanol; the first temperature is about 50° C.; the second temperature is about 27° C. or below; and the aqueous dialysis solution is initially comprised of water; and wherein after a period of dialysis against water, at least one of the soluble phase or the aqueous dialysis solution is adjusted to comprise about 0.88% formic acid.
 7. A method for obtaining heat stable proteinase inhibitor protein from potato tubers containing the same, comprising the steps of: extracting the potato tubers by homogenizing the potato tubers in a medium comprising about 0.2% formic acid and approximately 0.3 M NaCl to form crude extract; adding about one volume of ethanol to four volumes of crude extract to form an alcohol extract; filtering the alcohol extract to form a filtered extract; heating the filtered extract to a first temperature of 50° C. or higher followed by cooling to a second temperature of 50° C. or lower, to form a heat-treated extract containing an insoluble precipitate phase and a soluble phase; separating the insoluble precipitate phase from the soluble phase by at least one of, filtering the heat-treated extract through a porous filter, or centrifuging the heat-treated extract at about 4000× g for about 5 minutes, to form a clarified soluble phase; removing impurities from the clarified soluble phase by dialysis using a dialysis membrane having a molecular weight cutoff of approximately 12,000 daltons, against a dialysis medium comprising either tap water, or about 0.2% formic acid, to form a dialyzed extract; precipitating the proteinase inhibitor proteins from the dialyzed extract by at least one of; including about 0.22% formic acid in the dialysis medium, or adding formic acid to about 0.88%, to form a precipitation mixture; recovering the precipitated proteinase inhibitor proteins by at lease one of, filtering the precipitation mixture through a porous filter, or centrifuging the precipitation mixture at about 4000× g for about 5 minutes, to obtain a mixture of proteinase inhibitor protein.
 8. A heat stable proteinase inhibitor protein obtained according to the method of any one of claims 1-7. 